Apesar de bem esclarecidos os efeitos benéficos do exercício físico aeróbio na prevenção e tratamento de doenças cardiovasculares, muitos dos mecanismos de ação ainda permanecem desconhecidos. Mais especificamente relacionado à aterosclerose, vários trabalhos mostraram a eficiência de diferentes modalidades (esteira ou natação) de treinamento físico na redução ou prevenção do desenvolvimento da placa aterosclerótica. Este efeito é associado à redução de marcadores inflamatórios ou EROs, ou ainda ao aumento na produção e biodisponibilidade de NO e de enzimas antioxidantes, benefícios também observados para outras doenças cardio-metabólicas associadas com diferentes fatores de risco. Porém, pouco se sabe sobre o possível papel do exercício físico aeróbio na prevenção da doença aterosclerótica associada à hipercolesterolemia causada pela disfunção do LDLr, de fundamental importância para pacientes com HF.
A disfunção endotelial que começa a aparecer por volta do estágio II do desenvolvimento da aterosclerose, ou seja, antes do desenvolvimento do ateroma, é caracterizada por redução do relaxamento dependente do endotélio à acetilcolina. Sendo assim, uma das propostas do presente trabalho foi avaliar o efeito do treinamento físico aeróbio realizado em esteira rolante na prevenção da disfunção endotelial em camundongos LDLr-/- modelo experimental de HF, alimentados com dieta padrão. Para tal, o protocolo de treinamento foi iniciado nos animais com 12 semanas de vida, que representa um período no qual a placa aterosclerótica é mínima ao longo da aorta neste modelo experimental(Lichtman et al. 1999; Ma et al. 2012; Wrobel et al. 2013).
De forma inovadora avaliamos também o efeito da interferência do exercício físico aeróbio na expressão proteica da nNOS e na produção de H2O2 em
aorta de camundongos hipercolesterolêmicos. Esse mecanismo destaca-se devido a propriedade antiaterogênica récem-demonstrada para o H2O2 (Langbein et al.
2015a; Gray et al. 2016), assim como a nNOS como fonte de produção endotelial deste fator (Capettini et al. 2011). Embora tenha sido sugerida a participação da nNOS e do H2O2 na disfunção endotelial associada à aterosclerose em modelos
experimentais, não existem trabalhos na literatura que mostrem os possíveis efeitos benéficos do treinamento físico nesta via.
Em suma, no presente trabalho levantamos a hipótese de que o exercício físico aeróbio poderia ser preventivo na disfunção endotelial que precede o desenvolvimento da doença aterosclerótica, por aumentar a produção de NO e H2O2
concomitante com redução dos níveis de ânion superóxido, que em conjunto caracterizariam uma melhora do equilíbrio redox em aorta de camundongos LDLr-/- hipercolesterolêmicos.
3. OBJETIVO
Investigar os efeitos vasculares de um programa de treinamento físico aeróbio em camundongos LDLr-/-, modelo de hipercolesterolemia genética, com ênfase nos mecanismos associados à produção de NO, H2O2 e ânion superóxido.
4. ARTIGO
“Aerobic exercise training protects against endothelial dysfunction by increasing nitric oxide and hydrogen peroxide production in LDL receptor-
deficient mice”
Daniele M Guizoni, Gabriel G Dorighello, Helena C F Oliveira, Maria A Delbin, Marta H Krieger, Ana P Davel
Department of Structural and Functional Biology, Institute of Biology, University of Campinas - UNICAMP, Campinas, Sao Paulo, Brazil
Running title: Running training improves endothelial function in LDLr-/- mice
Corresponding author: Ana P. Davel, PhD Department of Structural and Functional Biology Institute of Biology P.O. Box 6109 University of Campinas -UNICAMP Campinas, SP, Brazil Telephone: +55 19 35216189
Fax: +55 19 35216185 E-mail: anadavel@unicamp.br
Abstract
Background: Endothelial dysfunction associated with hypercholesterolemia is an early event in atherosclerosis characterized by redox imbalance associated with high superoxide production and reduced nitric oxide (NO) and hydrogen peroxide (H2O2)
production. Aerobic exercise training (Ex) has been demonstrated to ameliorate atherosclerotic lesions and oxidative stress in advanced atherosclerosis. However, whether Ex protects against the early mechanisms of endothelial dysfunction in familial hypercholesterolemia remains unclear. This study investigated the effects of Ex on endothelial dysfunction and vascular redox status in the aortas of LDL receptor knockout mice (LDLr-/-), a genetic model of familial hypercholesterolemia.
Methods: Twelve-week-old C57BL/6J (WT) and LDLr-/- mice were divided into sedentary and exercised (Ex on a treadmill 1 h/5× per week) groups for 4 weeks. Changes in lipid profiles, endothelial function, and aortic NO, H2O2 and superoxide
production were examined.
Results: Total cholesterol and triglycerides were increased in sedentary and exercised LDLr-/- mice. Endothelium-dependent relaxation induced by acetylcholine was impaired in aortas of sedentary LDLr-/- mice but not in the exercised group. Inhibition of NO synthase (NOS) activity or H2O2 decomposition by catalase
abolished the differences in the acetylcholine response between the animals. No changes were noted in the relaxation response induced by NO donor sodium nitroprusside or H2O2. Neuronal NOS expression and endothelial NOS
phosphorylation (Ser1177), as well as NO and H2O2 production, were reduced in
aortas of sedentary LDLr-/- mice and restored by Ex. Incubation with apocynin increased acetylcholine-induced relaxation in sedentary, but not exercised LDLr-/- mice, suggesting a minor participation of NADPH oxidase in the endothelium- dependent relaxation after Ex. Consistent with these findings, Nox2 expression and superoxide production were reduced in the aortas of exercised compared to sedentary LDLr-/- mice. Furthermore, the aortas of sedentary LDLr-/- mice showed reduced expression of superoxide dismutase (SOD) isoforms and minor participation of Cu/Zn-dependent SODs in acetylcholine-induced, endothelium-dependent relaxation, abnormalities that were partially attenuated in exercised LDLr-/- mice. Conclusion: The data gathered by this study suggest aerobic physical training as a potential non-pharmacological therapy in the prevention of very early endothelial dysfunction and redox imbalance in familial hypercholesterolemia via increases in NO bioavailability and H2O2 production.
Keywords: Aerobic exercise training; LDL receptor-deficient mice; familial hypercholesterolemia; endothelial dysfunction; nitric oxide synthase; hydrogen peroxide; superoxide dismutase.
Introdution
Familial hypercholesterolemia (FH) is a genetic disorder characterized by high concentrations of low-density lipoprotein (LDL), resulting in premature development of atherosclerosis and its vascular complications [1]. Endothelial dysfunction has been described as an important early marker of atherosclerosis preceding atheroma formation and is characterized by impaired vasodilatation [2]. Therefore, non-pharmacological strategies preventing the onset of endothelial dysfunction may protect against the acceleration of atherosclerosis in FH.
LDL receptor knockout (LDLr-/-) mice are a genetic model of hypercholesterolemia that mimics human FH [3]. It is accepted that LDLr-/- mice fed a chow diet develop small lesions in the aortic root and impaired endothelium- dependent relaxation in the thoracic aorta [4,5], despite lacking atherosclerotic plaques throughout the descending aorta [6-8]. In this model of genetic hypercholesterolemia, endothelial dysfunction is associated with impaired hydrogen peroxide (H2O2) production [4]. Similar to the effects of endothelial NOS (eNOS)-
derived nitric oxide (NO), H2O2 production by neuronal nitric oxide synthase (nNOS)
contributes to endothelium-dependent relaxation in the mouse aorta [9]. Furthermore, increased NADPH oxidase Nox2-derived superoxide anion production, together with a reduction in NO bioavailability, is often found to contribute to the development of atherosclerosis in humans and experimental models [10-12]. Thus, reactive oxygen species (ROS) participate in the pathogenesis of atherosclerotic disease via multiple pathways.
It is well established that aerobic exercise training promotes vascular beneficial effects, mitigating the risk of cardiovascular disease [2]. Aerobic exercise training has been demonstrated to improve oxidative stress and endothelium- dependent relaxation, delaying the progression of vascular injury in atherosclerosis- prone model apolipoprotein E knockout mice (ApoE-/-) fed a high-fat and cholesterol diet [11,13]. Atherogenic diets are often associated with metabolic disorders, including obesity and type II diabetes. Previous studies have demonstrated that treadmill training improves endothelial dependent-relaxation [14], oxidative stress [15], NO release and nNOS expression [16] in obese non-atherosclerosis-prone rats. However, the potential beneficial effects of aerobic exercise training in the early
stages of atherosclerosis, independent of atherogenic diet-induced confounding factors, are unclear. Recently, Langbein et al. (2015) [5] demonstrated that voluntary physical activity was not able to improve endothelial dysfunction in aortas from LDLr-/- mice fed a standard or high-fat diet. However, it is still unknown whether or not an aerobic exercise training program can modulate endothelial function and vascular redox status in aortas from LDLr-/- mice.
Therefore, in the present study, we investigated the effect of treadmill training on impaired endothelium-induced relaxation in LDLr-/- mice, focusing on the effects of vascular production of the superoxide anion, NO, and H2O2.